The basis for the emergence of mefE-associated macrolide resistance in Streptococcus pneumoniae is a genetic element, mega (macrolide efflux genetic assembly). Mega initially emerged in pneumococcal serotypes (e.g., 14, 6B, 9V, 19F and 6A) and has now appeared in serotypes (e.g., 33F, 19A) causing replacement carriage and disease. We identified and characterized the mega genetic locus in S. pneumoniae. The mefE gene is found in single copy on the 5'end of a 5.5 kb or 5.4 kb chromosomal insert in the S. pneumoniae genome. At least four sites of insertion of mega have been identified. In addition to mefE, four other open reading frames (ORFs) organized into two convergent clusters are found in mega. Immediately downstream of mefE is an ORF, designated mel, that had significant homology to the erythromycin resistance ATP-binding cassette (ABC) protein, MsrA, of Staphylococcus epidermidis. The 3'end of mega contains three convergently transcribed ORFs (ORF5, ORF4, ORF3), homologous to ORFs in the 47.5 kb transposon Tn5252. In mefE-containing invasive S. pneumoniae isolates, mega is found in at least four distinct sites in the pneumococcal genome in epidemiologically and genetically unrelated strains. The gene products of mefE and mel are both required for macrolide resistance, work in concert, are coordinately regulated, and are inducible by macrolides. Mega element homologs have now been found in Tn1207.1, Tn1207.3, Tn2009 in S. pneumoniae and related elements in S. pyogenes, group B streptococci and in other Gram-positive bacteria. In two Specific Aims, we will further characterize the mega element and the genetic basis of mef-mediated macrolide resistance in S. pneumoniae. Two hypotheses will be examined: first, Mef and Mel of mega represent a novel efflux pump in Gram-positive bacteria composed of two distinct efflux proteins, an ABC membrane-bound transporter and a proton motive force transporter. Second, mega is a defective or "hitchhiker" genetic element related to conjugative transposons. The horizontal transfer and efflux activity of mega is enhanced by Tn5252 or other conjugative transposons. Also, the rapid emergence of mega and its continued spead in S. pneumoniae has been facilitated through ORFs 3-5 by competence induction and by agents, e.g., trimethoprim-sulfa, fluoroquinolones, that induce an SOS stress response in pneumococci. In Specific Aim 1, the molecular basis, regulation and substrate specificity of the efflux pump will be determined and the pump's potential role as a virulence factor assessed. In Specific Aim 2, the factors that impact the dissemination of mega (horizontal transfer) and efflux functions in the S. pneumoniae population will be determined. These studies should provide further insights into the molecular basis and mechanism(s) of dissemination of this novel genetic element and efflux pump and provide greater understanding of the emergence of antibiotic resistance in S. pneumoniae.